This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The oxidative stress hypothesis of aging postulates that senescence-associated attenuations in physiological function are caused by molecular oxidative damage induced by reactive oxygen species (ROS). Although there is considerable evidence implicating oxidative stress in aging, additional data are needed to clearly define the nature of this involvement. Ascorbate (AsA) is a major contributor to the antioxidant capacity cells have to counteract the action of ROS. Preliminary studies have shown significant signs of enhanced longevity, expanded reproductive period, and increased vigor in high-AsA lines of the Arabidopsis thaliana plant. These transgenics expressing genes of the myo-inositol (MI) pathway to AsA (a route that resembles the last steps of the animal pathway to AsA), and the genetic and genomic resources of Arabidopsis constitute a powerful system to explore the mechanisms and biological basis of an AsA-mediated resistance to aging, a subject of significant importance in biomedical research. Aims 1-3 will test the hypothesis that ascorbate is providing high-AsA Arabidopsis lines additional protection against oxidative stress, and that this protection is critical for the increased lifespan and extended reproductive activity displayed by those plants. In Aim 1, the role of AsA in modulating the temporal control of life-history traits [growth, lifespan, length of reproductive activity, and senescence] will be characterized. In Aim 2, the route(s) leading to AsA formation linked to the extended longevity phenotype will be determined. We propose leveraging the repertoire of knockout mutants we have developed for specific AsA biosynthetic enzymes to learn if there is a pathway linked to this phenotype. It is known that in Arabidopsis, somatic tissue longevity is not governed by reproductive development. In Aim 3, the route(s) leading to AsA formation linked to the extended flowering phenotype will be determined. Reproductive and actively growing tissues have higher metabolic rates and, therefore, a higher demand for antioxidants. In Aim 4, the cellular and subcellular localization of enzymes of the MI pathway to AsA (MI oxygenase [MIOX], glucuronate reductase, glucuronolactonase and gulono-1,4-lactone oxidase [GLOase]) will be determined in order to learn about the contribution of the different isoforms to the AsA pool in various tissues and organelles. A deeper and better understanding of the antioxidant activities in cells will contribute to developing therapies to combat/retard some of the destructive processes associated with aging, processes common to plants, animals, and humans.